Ec9706

Human ESCC cell lines (KYSE-140, KYSE-30, EC9706, TE-10), and human normal esophageal epithelial cell line (HET-1A), from ATCC (Rockville, Maryland), were propagated in the DMEM (Invitrogen, Carlsbad, CA) under the standard condition of 37°C and 5% CO₂. The 10% FBS and 1% Pen/Strep mixture, both from Invitrogen, served as supplements for DMEM.

Human normal esophageal epithelial cells (HET-1A) and EC cell lines (TE-1, TE-9, KYSE30, EC9706) were purchased from ATCC. Culture medium for EC cell lines: Dulbecco’s Modified Eagle Medium (DMEM, Hyclone) plus 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin solution (100X, Solarbio). The cells were incubated in a humidified incubator at 37° in with 5% CO2.
Before transfection, the medium was replaced with FBS-free medium. During transfection, 1 × 10⁵ cells were inoculated into each well of six-well plates. MiR-216a mimics (miR mimics), miR-216a inhibitor (miR inhibitor), NC mimics, NC inhibitor, HMGB3 siRNA, NC siRNA vectors were purchased from Sangon Bioengineering (Shanghai, China). The cell lines were transfected using the Lipofectamine 2000 kit (Invitrogen, USA).

Cell lines 293FT, EC9706, MCF-7, Hela, and HepG2 (ATCC) were maintained in Dulbecco’s MEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. HEK293FT cells were supplemented additionally with 1 mM L-glutamine. Jurkat and K562 cell lines (ATCC) were maintained in RPMI supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cell lines were cultured at 37°C with 5% CO₂. Cell lines were transfected with either Lipofectamine LTX (Invitrogen) or Nucleofector technology (Lonza) as per manufacturer’s instructions. For TALE activation experiments, we transfected all cell lines (150,000 cells per well of a 6-well plate) with the exception of Jurkat cells with 900 ng of empty vector control or TALE-encoding plasmids, in which each TALE plasmid was represented in equal amounts (e.g. two TALEs - 450 ng each or four TALEs –225 ng each) to assess minimal number of TALE activators required for gene activation. For experiments using Jurkat cells, 1x10⁶ cells were transfected with 2 µg of empty vector or TALE-encoding plasmids using equal amounts of each plasmid (e.g. two TALEs –1 µg each or four TALEs –500 ng each). Seventy-two hours post-transfection, cell culture media and cell pellets were collected and stored at −80°C for ELISA analyses and qRT-PCR, respectively.

ESCC cell lines, KYSE30, KYSE510, KYSE70, KYSE140, TE13, EC109, and EC9706, were purchased from ATCC and grown under 5% CO2 and 21% O2 in RPMI 1640 (PM150110, Pricella) including 10% FBS (S711-001s, Lonsera). Verification of the ESCC cell lines was conducted based on cellular morphology, and authentication was performed using STR DNA analysis. Additionally, tests were performed to confirm that there was no mycoplasma contamination in cells.

The paired tumor tissues and adjacent normal tissues (n = 42) were collected from ESCC patients who underwent surgery resection at the Shangqiu first People’s Hospital. This study was approved by the Ethics Committee of Shangqiu first People’s Hospital and informed consents were signed by all patients. The normalized RNA-seq data of Esophageal Carcinoma (ESCA) were downloaded from the TCGA data portal website (https://cancergenome.nih.gov/).
Human immortalized esophageal epithelial cell line HET-1A and human ESCC cell lines (ECA109 and EC9706) were purchased from ATCC (Manassas, VA, USA). All cells were cultured in PRMI-1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% FBS (Gibco) at 37 °C with 5% CO₂. DDP-resistant variants (ECA109/DDP and EC9706/DDP) of ECA109 and EC9706 cells were established using a repetitive pulsatile treatment with constant concentrations of cisplatin [17 (link)]. The degree of chemotherapy resistance of DDP-resistant variants was evaluated before transfections.

In this study, the human EC cell lines (Eca109, KYSE150, EC9706) and normal esophageal epithelial cells (Het-1A) were acquired from the ATCC (Manassas, VA, USA). These cells were cultured in 1640 medium (RPMI; Gibco, CA, USA) and supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco, CA, USA). HEK-293T cells were maintained in DMEM (Gibco, CA, USA) and added the same ingredients as above. These cells were cultured at 37°C under 5% CO2.